1. Field of the Invention
The present invention relates to a polypeptide capable of interacting with thrombin. More particularly, the present invention is concerned with a novel, substantially pure polypeptide with a low molecular weight, which interacts with thrombin to form a binding therebetween, thereby exhibiting an activity to inhibit the blood coagulation and platelet aggregation by thrombin and/or an activity to promote the thrombin-catalyzed activation of protein C. The present invention is also concerned with a DNA comprising a nucleotide sequence coding for the polypeptide; a recombinant DNA molecule comprising a replicable expression vector and the DNA; a transformant containing the recombinant DNA molecule; a process for producing the above-mentioned polypeptide; and a pharmaceutical composition comprising the polypeptide.
The polypeptide of the present invention participates in anticoagulation of blood and fibrinolysis with respect to blood coagulation, and has excellent activities of anticoagulation, platelet aggregation inhibition and thrombolysis. The polypeptide of the present invention is therefore useful as an active ingredient of drugs, particularly those for the treatment of diseases involving undesired blood-coagulation, such as myocardial infarction, thrombosis, embolism, obstruction of peripheral blood vessels, arteriosclerosis obliterans, disseminated intravascular coagulation (DIC) syndrome, angina pectoris, transient ischemic attack and toxemia of pregnancy.
In the present specification, amino acids and peptides are represented using abbreviations, as indicated below, approved by the IUPAC-IUB Commission on Biochemical Nomenclature (CBN). With respect to amino acids and the like having isomers, those represented by the following abbreviations are of the L-configuration unless otherwise specified. Further, unless otherwise specified, the left end and right end of the amino acid sequences of peptides are the N-terminus and C-terminus, respectively.
Gln: glutamine residue PA1 Asp: Aspartic acid residue PA1 Pro: proline residue PA1 Tyr: tyrosine residue PA1 Val: valine residue PA1 Lys: lysine residue PA1 Glu: glutamic acid residue PA1 Ala: alanine residue PA1 Asn: asparagine residue PA1 Leu: leucine residue PA1 Phe: phenylalanine residue PA1 Gly: glycine residue PA1 His: histidine residue PA1 Ser: serine residue PA1 Thr: threonine residue PA1 Ile: isoleucine residue PA1 Trp: tryptophan residue PA1 Arg: arginine residue PA1 Met: methionine residue PA1 Cys: cysteine residue PA1 A: 2'-deoxyadenylic acid residue PA1 C: 2'-deoxycytidylic acid residue PA1 G: 2'-deoxyguanylic acid residue PA1 T: thymidylic acid residue
Polydeoxyribonucleotides and oligodeoxyribonucleotides are represented by sequences of deoxynucleotide residues which are abbreviated as follows:
Unless otherwise specified, the left end and the right end of the sequence of deoxynucleotides are the 5' end and 3'end, respectively.
2. Discussion of Related Art
Protein C is known as a protein which is dependent on vitamin K and which plays an important role in the blood coagulation mechanism. In recent years, it has been reported that a substance which interacts with thrombin to form a binding therebetween, thereby exhibiting activities to repress the platelet activation and fibrin formation by the action of thrombin and promote the thrombin-catalyzed activation of protein C, is present in rabbit lung, bovine lung, human lung, human placenta and the like. Such a substance is generally called "thrombomodulin".
N. L. Esmon et al. (see J. Biol. Chem., vol. 257, pages 859-864, 1982) reported that a substance separated from rabbit lung and purified requires calcium ions at the time of activating protein C after forming a bond to thrombin. Further, K. Suzuki et al. (see Biochemica et Biophysica Acta, vol. 882, pages 343-352, 1986) reported that a substance separated from bovine lung and purified also requires calcium ions at the above-mentioned time, and this requirement for calcium ions with respect to a substance separated from human placenta and purified was reported by H. H. Salem et al. (see J. Biol. Chem., vol. 259, pages 12246-12251, 1984). It was reported by S. Kurosawa et al. (see J. Biol. Chem., vol. 262, pages 2206-2212, 1987) that a soluble peptide, obtained by digesting with elastase the above-mentioned substance separated from rabbit lung and purified, exhibits the highest activity at a calcium ion concentration of 0.3 mM in the activation of protein C, and that such a dependence on calcium ion concentration is not observed at the time of the activation of protein C having the Gla domain deleted (such protein C hereinafter referred to as "GDPC").
S. Yamamoto, who is one of the present inventors, and his colleagues have disclosed the cloning of a human-derived thrombomodulin cDNA (see International Application Publication No. W088/05053).
In accordance with the recent progress of genetic engineering, it is now possible to replace one or more predetermined amino acid residues in a protein with other amino acid residues and also to delete an amino acid sequence from a predetermined site of a protein. Various researches have been made to create a novel protein meeting a particular objective by modifying a natural protein using genetic engineering. With respect to human thrombomodulin, the present inventors (see M. Zushi et al., J. Biol. Chem., vol. 264, pages 10351-10353, 1989) have shown that a polypeptide comprised of 115 amino acid residues has an ability to promote the thrombin-catalyzed activation of protein C.
In the treatment of diseases involving undesired blood coagulation, the administration of a pharmaceutical composition comprising a polypeptide is mainly effected by intravenous drip or local administration by PTCR (percutaneous transluminal coronary recanalization). With respect to the administration of human thrombomodulin, intravenous drip is preferred to local administration by PTCR. Such an administration method is acceptable for a short-term treatment, but it causes grave mental and economic burden on patients who receive a long-term treatment or preventive medication. Further, because the molecular weight of conventional polypeptides is generally large and only a limited portion of the polypeptide chain contributes toward pharmaceutical activity, the dose of a pharmaceutical composition comprising a polypeptide is inevitably large, and this is likely to create a danger of patient antigenecity.